Use of homosalate and octyl salicylate to treat multiple sclerosis

ABSTRACT

A method of treating an MS patient with homosalate, octyl salicylate, or a combination is disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/643,988 filed on Jul. 7, 2017 which claims benefit from U.S.Provisional Application 62/362,951, filed Jul. 15, 2016, the contents ofwhich are incorporated herein by reference for all purposes.

BACKGROUND OF THE INVENTION

Multiple sclerosis (MS), the most common autoimmune disorder affectingthe central nervous system, is a demyelinating disease in which theinsulating covers of nerve cells in the brain and spinal cord aredamaged. The disease affects the ability of parts of the nervous systemto communicate and results in physical, mental, and sometimespsychiatric problems. For example, MS patients often have visiondisorders, such as blindness in one eye or double vision. Patients alsoexhibit muscle weakness, trouble with sensation, and trouble withcoordination.

The underlying mechanism of MS progression is thought to be eitherdestruction by the immune system or failure of the myelin-producingcells. The cause of the disease is not known, but may include geneticsand environmental factors such as viral infections. MS is usuallydiagnosed based on analysis of a patient's symptoms and the results ofsupporting medical tests.

Unfortunately, there is no known cure for multiple sclerosis. Currenttreatments attempt to improve symptoms after an attack and prevent newattacks. Medications used to treat MS are only modestly effective andcan have side effects and be poorly tolerated. Physical therapy can helpwith a patient's ability to function. Because of the lack of goodalternatives, many patients pursue unproven alternative treatments. Lifeexpectancy is 5 to 10 years lower than that of an unaffectedpopulation.^([2])

Needed in the art are new therapeutic compounds for effective MStreatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 discloses experiments showing that topical application ofsunscreen protects mice from EAE progression. Mice received topicalapplication of sunscreen, NBUVB or both. Each mouse was scored dailyafter EAE during the entire experiment (day 0-day 28) and data areexpressed as mean value. N=10-12/group.

FIGS. 2A and 2B are a set of graphs showing the effect of specificsunscreen ingredients on EAE. Ingredients (avobenzone and oxybenzone) ofsunscreen partially suppress EAE severity in mice. FIG. 2A: Analysis ofdifferent sunscreen ingredients. Avobenzone and oxybenzone partiallysuppress EAE severity in mice. Mice were treated with sunscreeningredients (avobenzone, oxybenzone or combination) by topicaladministration daily during EAE (day 0-day 30). Sunscreen spray was usedas a positive control. Each mouse was scored daily and data areexpressed as mean values. N=10-12/group. FIG. 2B: Body weights of micethat were administered different sunscreen ingredients. Mice weretreated with sunscreen ingredients (avobenzone, oxybenzone orcombination) by topical administration daily during EAE (day 0-day 30).Sunscreen spray was also used as a positive control. Each mouse wasweighed weekly during the experiment and data are expressed as meanvalue. N=10-12/group.

FIGS. 3A and 3B are a set of graphs showing the effect of sunscreeningredients on EAE. FIG. 3A: Sunscreen ingredients (homosalate and octylsalicylate) dramatically suppress EAE development in mice. Mice weretreated with sunscreen ingredients (homosalate, octyl salicylate orcombination) by topical administration daily during EAE (day 0-day 30).Sunscreen spray was used as a positive control. Mice were scored dailyand data are expressed as mean value. N=1-12/group. FIG. 3B: Bodyweights of mice administered different sunscreen ingredients. Mice weretreated with sunscreen ingredients (homosalate, octyl salicylate orcombination) by topical administration daily during EAE (day 0-day 30).Sunscreen spray was used as a positive control. Mice were weighed weeklyand data are expressed as mean value. N=11-12/group.

FIGS. 4A, 4B, and 4C show that topical application of COPPERTONE SPRAYsunscreen (SS) completely blocks EAE. FIG. 4A: Topical application ofsunscreen prior to NBUVB. Mean score was recorded daily after inductionof EAE. FIG. 4B: Body weights were measured weekly. FIG. 4C: Topicalapplication of sunscreen with or without NBUVB completely blocked EAEdevelopment. Mean score was measured daily after immunization. Data areexpressed as mean±SEM in FIGS. 4A and 4C; Data are expressed as mean±SDin FIG. 4B. All treatment groups in FIGS. 4A, 4B and 4C werestatistically different from control group (n=12, p<0.05).

FIGS. 5A and 5B show that commercial sunscreen preparationsdifferentially block EAE while darkness itself does not. FIG. 5A: Sixdifferent sunscreens were topically administered daily and mean scorerecorded. FIG. 5B: The effect of total darkness on EAE was determined.Treated mice were kept in total darkness for various periods as shown inthe legend. Mean score was measured daily. Data are expressed as meanvalues in FIGS. 5A and 5B. All treatment groups in panel A except BANANABOAT KIDS and CoTZ FACE were statistically different from control (n=12,P<0.05). No statistical differences were found amongst the groups inpanel B (n=12).

FIGS. 6A, 6B, 6C and 6D show that the suppression of EAE by COPPERTONESPRAY sunscreen (SS) is both time and dose dependent. FIG. 6A: Diseasescores in relation to time of administration of Coppertone Spraysunscreen. Mean score was determined daily. Mice were treated topicallywith SS 200 μl daily for entire experiment (day 30) with variousstarting treatment time. Pretreatment: treatment started at day −7before immunization; Immunization: treatment started at time ofimmunization; onset: treatment initiated when animals first exhibited ascore ≥1.0. FIG. 6B: Body weights determined each week. FIG. 6C:Dose-dependent suppression of EAE by Coppertone Spray sunscreen. FIG.6D: Body weight was determined weekly. Data are expressed as mean±SEM inFIGS. 6A & 6C; mean±SD in FIGS. 6B & 6D. Pretreatment and Immunizationtreatments were significantly different from control group in panels Aand B (n=12, p<0.05). The two highest levels of SS (50 and 100 μl) weresignificantly different from control in panels C&D (n=12, p<0.05).

FIGS. 7A, 7B, 7C and 7D show that two ingredients of effective sunscreen(Homosalate and octisalate) significantly suppress EAE. Two otheringredients (avobenzone and oxybenzone) fail to suppress EAE. FIGS. 7Aand 7B: Mice were treated with a solution of (12% avobenzone, 16%oxybenzone or combination in 25 μl of DMSO) topically each day.Sunscreen (Coppertone spray) (100 μl) was used as a positive control.Each mouse was scored daily (FIG. 7A) and weighed weekly (FIG. 7B)during the experiment. FIGS. 7C and 7D: Mice were treated with twoingredients (30 μl homosalate (1.5 g/kg), 10 μl octisalate (0.5 g/kg)and combination) topically. COPPERTONE SPRAY sunscreen 200 μl was usedas a positive control. Mean score (FIG. 7C) and body weight (FIG. 7D)were recorded. Data are expressed as mean value. In FIGS. 7A & 7B, allgroups were significantly different from the sunscreen group (n=12,p<0.05). In FIGS. 7C and 7D, all treatment groups except octisalatealone were significantly different from the control groups (p<0.05).

FIGS. 8A, 8B, 8C and 8D show dose dependent suppression of EAE byhomosalate (HS) and octisalate (OS). FIGS. 8A and 8B: Mice were treatedwith various doses (10, 20 and 30 μl or 0.5, 1.0, 1.5 g/kg) ofhomosalate or octisalate topically. The mean score (FIG. 8A) and bodyweights (FIG. 8B) were recorded. FIGS. 8C and 8D, Mice treated withhomosalate (30 μl or 1.5 g/kg) topically at various times. Daily meanscore (FIG. 8C) and body weight (FIG. 8D) were recorded. Data areexpressed as mean value. In FIG. 8A, all treatment groups weresignificantly different from control (p<0.05). In FIG. 8B, alltreatments except homosalate 0.5 g/kg were significantly different fromcontrol (p<0.05); In FIGS. 8C and 8D, the mean scores of homosalateevery day and every 2-day were significantly lower than control(p<0.05).

FIGS. 9A, 9B, and 9C show that all of the tested sunscreens protect themice from developing EAE.

FIGS. 10A, 10B, 10C, 10D, 10E and 10F show that all of the testedsunscreens protect the mice from developing EAE.

DESCRIPTION OF THE PREFERRED EMBODIMENT Terms

The terms “including” and “comprising” are open-ended terms and shouldbe interpreted to mean “including, but not limited to . . . .” Theseterms encompass the more restrictive terms “consisting essentially ofand “consisting of.”

The singular forms “a”, “an”, and “the” include plural reference. Aswell, the terms “a” (or “an”), “one or more” and “at least one” can beused interchangeably. The terms “comprising”, “including”,“characterized by” and “having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Each of the publications andpatent documents specifically mentioned herein is incorporated byreference in its entirety for ail purposes including describing anddisclosing the chemicals, instruments, statistical analyses andmethodologies which are reported in the publications and which might beused in connection with the invention. All references cited in thisspecification are to be taken as indicative of the level of skill in theart. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

The “effective amount,” as used herein, refers to the amount of arequired to ameliorate the symptoms of a disease relative to anuntreated patient. The effective amount of active compound(s) used topractice the present invention for therapeutic treatment of a diseasevaries depending upon the manner of administration, the age, bodyweight, and general health of the subject. Ultimately, the attendingphysician or veterinarian will decide the appropriate amount and dosageregimen. Such amount is referred to as an “effective” amount.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. Unlessotherwise clear from context, all numerical values provided herein aremodified by the term “about.”

The term “multiple sclerosis” or “MS,” as used herein, refers to aninflammatory disease affecting the nervous system, in which the myelinsheaths around the axons of the brain and spinal cord are damaged,leading to demyelination and scarring as well as a broad spectrum ofclinical signs and symptoms. MS may be classified into different diseasesubtypes, including relapsing/remitting MS (RRMS), secondary progressiveMS, primary progressive MS, and progressive relapsing MS. Therelapsing-remitting subtype may be characterized by unpredictablerelapses followed by periods of months to years of relative quiet(remission) with no new signs of disease activity. Therelapsing-remitting subtype may usually begin with a clinically isolatedsyndrome (CIS). In CIS, a patient may have an attack suggestive ofdemyelination. Often CIS marks the onset of MS.

A diagnosis of multiple sclerosis can be established on the basis ofestablished clinical symptoms and the clinical symptoms is well known tothe skilled person. In one embodiment, the present invention disclose acomposition for treating MS by lessening occurrence of the symptom or byslowing or stalling progression of the symptom.

The clinical symptoms of multiple sclerosis may include vision problems,dizziness, vertigo, sensory dysfunction, weakness, problems withcoordination, loss of balance, fatigue, pain, neurocognitive deficits,mental health deficits, bladder dysfunction, bowel dysfunction, sexualdysfunction, heat sensitivity.

The term “multiple sclerosis” also refers to any other autoimmunedisease manifested by demylination of the central nervous system'sneurons. The first symptoms which appear at the onset of MS may bereferred to at times as “MS-related symptoms.” The symptoms of MS inEAE-induced animals (animal model of MS) may be typically weakness andmalfunction in the animal's tail, followed by weakness of its rear feetand finally weakness in its front feet. In humans, such first MS-relatedsymptoms may typically be double vision, facial numbness, facialweakness, vertigo, nausea, vomiting ataxia, weakness of the arms, etc.

The term “treating” or “treatment,” as used herein, refers toamelioration of some of the undesired symptoms of multiple sclerosis,the prevention of the manifestation of such symptoms before they occur,slowing down or completely preventing the progression of the disease (asmay be evident by longer periods between reoccurrence episodes, slowingdown or prevention of the deterioration of symptoms etc.), enhancing theonset of the remission period, slowing down the irreversible damagecaused in the progressive-chronic stage of the disease (both in theprimary and secondary stages), delaying the onset of said progressivestage, or a combination of two or more of the above.

The term “patient” or “subject,” as used herein, refers to a mammaliansubject (primates (e.g., humans), cows, sheep, goats, pigs, horses,dogs, cats, rabbits, rats, mice and the like), preferably a humansubject, that has, is suspected of having, or is or may be susceptibleto a condition associated with multiple sclerosis. In one embodiment,the present method may be used for treating a patient who suffers frommultiple sclerosis, e.g., with any symptoms as discussed above. Inanother embodiment, the present method may also be used to prevent aperspective patient from getting multiple sclerosis.

The term “perspective patient,” as used herein, refers to any person orsubject who may be or is in danger of developing MS.

The term “diagnosing” or “diagnosis,” as used herein, refers todetecting a disease or disorder or determining the stage or degree of adisease or disorder such as MS. The term “diagnosis” also encompassesdetermining the therapeutic effect of a drug therapy to treat MS, orpredicting the pattern of response to a drug therapy of MS. Thediagnostic methods may be used independently, or in combination withother diagnostic and/or staging methods known in the medical art for aparticular disease or disorder, e.g., MS.

The term “pharmaceutically acceptable,” as used herein, refers to thecompound or composition or carrier being suitable for administration toa subject to achieve the treatments described herein, without undulydeleterious side effects in light of the necessity of the treatment.

The term “therapeutically effective amount” or “pharmaceuticallyappropriate dosage,” as used herein, refers to the amount of thecompounds or dosages that will elicit the biological or medical responseof a subject, tissue or cell that is being sought by the researcher,veterinarian, medical doctor or other clinician.

As used herein, “pharmaceutically-acceptable carrier” includes any andall dry powder, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic agents, absorption delaying agents, and thelike. Pharmaceutically-acceptable carriers are materials, useful for thepurpose of administering the compounds in the method of the presentinvention, which are preferably non-toxic, and may be solid, liquid, orgaseous materials, which are otherwise inert and pharmaceuticallyacceptable, and are compatible with the compounds of the presentinvention. Examples may include sugars such as, but not limited to,lactose, glucose and sucrose; starches such as, but not limited to, cornstarch and potato starch; cellulose and its derivatives such as, but notlimited to, sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipientssuch as, but not limited to, cocoa butter and suppository waxes; oilssuch as, but not limited to, peanut oil, cottonseed oil, safflower oil,sesame oil, olive oil, corn oil and soybean oil; glycols; such apropylene glycol; esters such as, but not limited to, ethyl oleate andethyl laurate; agar; buffering agents such as, but not limited to,magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol, and phosphatebuffer solutions, as well as other non-toxic compatible lubricants suchas, but not limited to, sodium lauryl sulfate and magnesium stearate, aswell as coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants may alsobe present in the composition. Further examples of such carriersinclude, various lactose, mannitol, oils such as corn oil, buffers suchas PBS, saline, polyethylene glycol, glycerin, polypropylene glycol,dimethylsulfoxide, an amide such as dimethylacetamide, a protein such asalbumin, and a detergent such as Tween 80, mono- andoligopolysaccharides such as glucose, lactose, cyclodextrins and starch.

The term “administering” or “administration,” as used herein, refers toproviding the compound or pharmaceutical composition of the invention toa subject suffering from or at risk of the diseases or conditions to betreated or prevented.

The term “systemic delivery,” as used herein, refers to any suitableadministration methods which may delivery the compounds in the presentinvention systemically. In one embodiment, systemic delivery may beselected from the group consisting of oral, parenteral, intranasal,inhaler, sublingual, rectal, intracisternal, and transdermal,intravaginal, intraperitoneal, topically (as by powders, ointments ordrops), bucal or as an oral or nasal spray administrations. The term“parenterally” as used herein, refers to modes of administration whichinclude intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intraarticular injection and infusion.

In one embodiment, the present compositions or formulations may beadministered topically.

The term “topical administration,” as used herein, refers to localadministration of a component of a composition or a kit of the inventiononto the surface of a skin or mucosal tissue of a subject. A topicaladministration emphasizes local effect, and substance is applieddirectly where its action is desired. Sometimes, however, the termtopical may be defined as applied to a localized area of the body or tothe surface of a body part, without necessarily involving target effectof the substance, making the classification rather a variant of theclassification based on application location.

Methods of the Present Invention

The present invention includes methods of using octyl salicylate and/orhomosalate, or a composition comprising at least one of octyl salicylateand homosalate, to prevent or suppress the progression of multiplesclerosis. The Examples below show that Applicants observed thatcommercial sun block preparations decreased or prevented the incidenceof EAE in EAE mice, a mouse model of MS. Specifically, Applicantsdemonstrate that treating a multiple sclerosis (MS) patient or aprospective MS patient with an effective amount of a compositionselected from the group consisting of homosalate, octyl salicylate andcombinations of homosalate and octyl salicylate can prevent thedevelopment of experimental autoimmune encephalomyelitis, thus treatingMS. In contrast to the use of sunscreens that comprise octyl salicylateand homosalate, the method of the present invention requires the use ofthe composition daily for at least 30 days and preferably at least 60 or90 days. Additionally, a preferable form of administration of thepresent invention is orally, by inhalation or parenterally, in contrastto topical sunscreen administration.

The method of the present invention includes the step of diagnosing agroup of subjects (e.g., humans) in danger of developing MS andidentifying an MS patient or a perspective MS patient exhibiting atleast one symptom of MS.

Identification of a subject or patient appropriate for treatment of MSsymptoms can be carried out based on standardized diagnostic criteriawidely used by practicing physicians, especially in the first stages ofthe disease, such as the so-called Schumacher and Poser criteria(Compston A, Coles A, October 2008, Multiple sclerosis. Lancet 372(9648): 1502-17; Trojano M, Paoliceili D, (2001) The differentialdiagnosis of multiple sclerosis: classification and clinical features ofrelapsing and progressive neurological syndromes. Neurol, Sci. 22 (Suppl2): S98-102; Poser C M, Brinar V V (2004) Diagnostic criteria formultiple sclerosis: an historical review. Clin Neurol Neurosurg 108 (3):147-58), or the McDonald criteria, which focus on a demonstration withclinical, laboratory and radiologic data of the dissemination of MSlesions in time and space (Compston A, Coles A, October 2008, Multiplesclerosis. Lancet 372 (9648): 1502-17; McDonald W I, Compston A, Edan Get al, (2001) Recommended diagnostic criteria for multiple sclerosis:guidelines from the International Panel on the diagnosis of multiplesclerosis. Ann. Neurol. 50 (1): 121-7; Polman C H, Reingold S C, Edan Get al., (2005) Diagnostic criteria for multiple sclerosis: 2005revisions to the “McDonald Criteria”. Ann. Neurol, 58 (6): 840-6).

The most commonly used diagnostic tools for MS are neuroimaging,analysis of cerebrospinal fluid and evoked potentials. In a positivediagnosis, magnetic resonance imaging (MRI) of the brain and spine showsareas of demyelination (lesions or plaques). Gadolinium administered, asa contrast agent, to a patient with MS typically localizes in these “hotspots” or lesions, and can be easily identified with the use of MRI. TheMRI of the lesions is one of the most efficient methods of diagnosingMS. Measuring the development of new lesions is also a critical andefficient method of monitoring the progression of MS.

Alternatively, MS can be diagnosed with other known methods. Forinstance, an MS patient may respond less actively to stimulation of theoptic nerve (which may be examined using visual and sensory evokedpotentials) and sensory nerves due to demyelination of these nervepathways (Gronseth G S, Ashman E J, (2000) Practice parameter: theusefulness of evoked potentials in identifying clinically silent lesionsin patients with suspected multiple sclerosis (an evidence-basedreview): Report of the Quality Standards Subcommittee of the AmericanAcademy of Neurology. Neurology 54 (9): 1720-5). Chronic inflammation ofthe central nervous system can be demonstrated by an analysis ofcerebrospinal fluid. The cerebrospinal fluid is tested for oligoclonaibands, which are present in 75-85% of people with MS (McDonald W I,Compston A, Edan G et al, (2001) Recommended diagnostic criteria formultiple sclerosis: guidelines from the International Panel on thediagnosis of multiple sclerosis. Ann. Neurol. 50 (1): 121-7; Link H,Huang Y M, (2008) Oligoclonai bands in multiple sclerosis cerebrospinalfluid: an update on methodology and clinical usefulness. J.Neuroimmunol. 180 (1-2): 17-28).

In one embodiment, the method of diagnosing an MS patient or aperspective MS patient may include the steps of diagnosing oridentifying a subject with one or more of a delay of onset of MSsymptoms, with a reduction of peak of severity of MS symptoms, and/orwith a decrease of the cumulative disease index (CDI).

One would monitor the patient's MS symptoms and detect a reduction ordelay in these symptoms. Most preferably, the development of new lesionsin the subject would be monitored on a regular (i.e., semi-annual) basisvia MRI. Further symptoms that may be monitored include those selectedfrom the group consisting of changes in sensation (hypoesthesia andparaesthesia), muscle weakness, muscle spasms, or difficulty in moving;difficulties with coordination and balance (ataxia); problems in speech(dysarthria) or swallowing (dysphagia), visual problems (nystagmus,optic neuritis, or diplopia), fatigue, acute or chronic pain, andbladder and bowel difficulties. Cognitive impairment of varying degreesand emotional symptoms of depression or unstable mood are also common.One common clinical measure of disability progression and symptomseverity is the Expanded Disability Status Scale or EDSS.

In one embodiment, the patient's symptoms may include vision problems,dizziness, vertigo, sensory dysfunction, weakness, problems withcoordination, loss of balance, fatigue, pain, neurocognitive deficits,mental health deficits, bladder dysfunction, bowel dysfunction, sexualdysfunction, heat sensitivity, muscle weakness/numbness, and/orparalysis.

In one specific embodiment, the patient's symptom includes paralysis.

In another embodiment, the patient's symptom includes muscleweakness/numbness.

EAE (experimental autoimmune encephalomyelitis, sometimes experimentalallergic encephalomyelitis) is an animal model of brain inflammation anddemyelinating disease of the central nervous system (CNS) that has beensuccessful in developing useful therapeutic agents. The mouse model iswidely studied as an animal model of the human CNS demyelinatingdiseases, including multiple sclerosis and acute disseminatedencephalomyelitis (ADEM).

After comparing the ingredients found in the sun blocks, Applicantsidentified a few possible differences in the ingredients and thennarrowed in on four active ingredients in the effective sun blocks:avobenzone, oxybenzone, homosalate and octyl salicylate. Applicantstreated EAE mice with each of these four compounds after induction ofthe disease and monitored progression of the disease. While avobenzoneand oxybenzone slightly suppressed disease progression, homosalate andoctyl salicylate almost completely prevented EAE development.

In one embodiment, the present invention is a method of treating an MSpatient, comprising the steps of identifying an MS patient or aprospective MS patient and treating the patient with an effective amountof a compound selected from the group consisting of octyl salicylate,homosalate and a mixture of octyl salicylate and homosalate, wherein MSsymptoms are decreased or eliminated. As disclosed below, the amount ofoctyl salicylate, homosalate, or a mixture of octyl salicylate andhomosalate is not the same as the amount that would be applied to anindividual using sunscreen.

In another embodiment, progression of disease symptoms is slowed orstalled.

In one embodiment, the step of identifying an MS patient or aprospective MS patient may include any methods of identification asdiscussed above and/or any other methods known to one skilled in theart.

For example, one can identify an MS patient or a prospective MS patientby monitoring one of the symptoms which are typical to an MS patient ora prospective MS patient.

In one embodiment, one can monitor one or more symptoms including visionproblems, dizziness, vertigo, sensory dysfunction, weakness, problemswith coordination, loss of balance, fatigue, pain, neurocognitivedeficits, mental health deficits, bladder dysfunction, boweldysfunction, sexual dysfunction, heat sensitivity, muscleweakness/numbness, and/or paralysis.

In one specific embodiment, the monitored symptom includes paralysis.

In one specific embodiment, the monitored symptom is paralysis.

In another embodiment, the monitored symptom includes muscleweakness/numbness.

In another embodiment, the monitored symptom is muscleweakness/numbness.

In one embodiment, the present application uses a composition or aformulation comprising at least one of octyl salicylate and homosalate,to prevent or suppress the progression of multiple sclerosis in apatient.

In one embodiment, the present application may include at least one ofoctyl salicylate and homosalate as the sole active compounds fortreating, preventing or suppressing the progression of multiplesclerosis in a patient.

In another embodiment, the present application may include at least oneof octyl salicylate and homosalate along with other active compounds fortreating, preventing or suppressing the progression of multiplesclerosis in a patient.

In one embodiment, the target patient may be a mammalian subject(primates (e.g., humans), cows, sheep, goats, pigs, horses, dogs, cats,rabbits, rats, mice and the like), or a human subject. In oneembodiment, the patient is preferably a human being.

In one embodiment, a patient of the human subject is suspected ofhaving, or is or may be susceptible to a condition associated withmultiple sclerosis.

In one embodiment, the present application includes the step ofidentifying an MS patient or a prospective MS patient. For example, anMS patient or a prospective MS patient for the present application maybe identified by monitoring one or more symptoms including visionproblems, dizziness, vertigo, sensory dysfunction, weakness, problemswith coordination, loss of balance, fatigue, pain, neurocognitivedeficits, mental health deficits, bladder dysfunction, boweldysfunction, sexual dysfunction, heat sensitivity, muscleweakness/numbness, and/or paralysis of the subject.

In one embodiment, the step of identifying an MS patient or aprospective MS patient is by monitoring the symptom of paralysis ormuscle weakness/numbness.

In one embodiment, the step of identifying an MS patient or aprospective MS patient is by monitoring the symptom of paralysis.

In one embodiment, the step of identifying an MS patient or aprospective MS patient is by monitoring the symptom of muscleweakness/numbness.

In one embodiment, one can continue monitoring the symptom of thepatient during the treatment process for the purpose of evaluation ofthe treatment. For example, a patient's symptom such as paralysis ormuscle weakness/numbness may be monitored during the treatment so thatthe patient's treatment can be evaluated. As an example, the success ofthe treatment can be shown when occurrence of MS symptom is lessened orprogression of the symptom is slowed or stalled after the treatment.

After an MS patient or a prospective MS patient is identified, the MSpatient or the prospective MS patient is treated with a therapeuticallyeffective amount of a composition or formulation comprising a substanceselected from the group consisting of homosalate, octyl salicylate andcombinations of homosalate and octyl salicylate.

In one embodiment, the substance selected from the group consisting ofhomosalate, octyl salicylate and combinations of homosalate and octylsalicylate is the only active substance used in the present application.Thus, the MS patient or the prospective MS patient is treated with atherapeutically effective amount of a substance selected from the groupconsisting of homosalate, octyl salicylate and combinations ofhomosalate and octyl salicylate.

In another embodiment, the substance selected from the group consistingof homosalate, octyl salicylate and combinations of homosalate and octylsalicylate may be used as one of the active substances. Thus, the MSpatient or the prospective MS patient is treated with a therapeuticallyeffective amount of a composition or formulation comprising a substanceselected from the group consisting of homosalate, octyl salicylate andcombinations of homosalate and octyl salicylate.

The composition or formulation may additionally include anypharmaceutically acceptable carrier, pharmaceutically acceptable sterileaqueous or nonaqueous solutions, dispersions, suspensions or emulsions,adjuvants such as preservatives, wetting agents, emulsifying agents anddispersing agents, or other necessary substances.

In one embodiment, the composition or formulation is either in soliddosage forms or liquid dosage forms.

Liquid dosage forms may include pharmaceutically acceptable emulsions,solutions, suspensions, syrups and elixirs. In addition to the activecompounds, the liquid dosage forms may contain inert diluents commonlyused in the art such as, for example, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide,oils (in particular, cottonseed, groundnut, corn, germ, olive, castorand sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty acid esters of sorbitan and mixtures thereof.

Solid dosage forms may include capsules, tablets, pills, powders, andgranules. In certain embodiments, solid dosage forms may contain from 1%to 95% (w/w) of a compound of the invention. In certain embodiments, thepresent compounds or pharmaceutically acceptable salts thereof, may bepresent in the solid dosage form in a range of from 5% to 70% (w/w). Insuch solid dosage forms, the active compound may be mixed with at leastone inert, pharmaceutically acceptable carrier, such as sodium citrateor dicalcium phosphate and/or a), fillers or extenders such as starches,lactose, sucrose, glucose, mannitol, and silicic acid; b) binders suchas carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,sucrose, and acacia; c) humectants such as glycerol; d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; e) solutionretarding agents such as paraffin; f) absorption accelerators such asquaternary ammonium compounds; g) wetting agents such as cetyl alcoholand glycerol monostearate; h) absorbents such as kaolin and bentoniteclay and i) lubricants such as talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate and mixturesthereof. In the case of capsules, tablets and pills, the dosage form mayalso comprise buffering agents.

Dosage forms for topical administration of the compounds and theformulation described herein include powders, sprays, ointments, andinhalants. The active compound may be mixed under sterile conditionswith a pharmaceutically acceptable carrier and any needed preservatives,buffers or propellants which may be required. Ophthalmic formulations,eye ointments, powders and solutions are also contemplated as beingwithin the scope of this invention.

A compound of the invention may also be administered in sustainedrelease forms or from sustained release drug delivery systems.

In one embodiment, the compounds, compositions and the formulations maybe administered by a method of oral, parenteral, intranasal, inhaler,sublingual, rectal, intracisternal, and transdermal, intravaginal,intraperitoneal, topically (as by powders, ointments or drops), bucal oras an oral or nasal spray administrations.

In one embodiment, the compounds, compositions and the formulations maybe administered by oral, parenteral or topical method.

In one embodiment, the compounds, compositions and the formulations maybe administered topically.

In some embodiments, the dose of the invention regarding homosalateand/or octisalate is at least about 0.1 g/kg per body weight. In otherembodiments, the dose of the invention regarding homosalate and/oroctisalate is at least about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45,0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.05, 1.1,1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13,13.5, 14, 14.5 or 15 g/kg per body weight. In further embodiments, thedose of the invention regarding homosalate and/or octisalate is betweenabout 0.5 to about 15 g/kg per body weight. In other embodiments, thedose of the invention regarding homosalate and/or octisalate is betweenabout 0.5-14, 1-10, 2-10, 1-8, 1-5 or 0.5-2 g/kg per body weight.

In one embodiment, homosalate and/or octisalate is administered as theonly active ingredient in a composition or formulation as discussedabove. For example, a composition of homosalate and/or octisalate may beadministered to a MS patient or a perspective patient. The compositionof homosalate and/or octisalate may be combined with other componentssuch as any pharmaceutically acceptable carrier, pharmaceuticallyacceptable sterile aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, adjuvants such as preservatives, wettingagents, emulsifying agents and dispersing agents, or other necessarysubstances.

In one embodiment, Applicants envision homosalate and/or octisalate maybe administered as one of the multiple active ingredients for treatingMS. For example, Applicants envision that homosalate and/or octisalatemay be used with other MS treatment protocols such as cyclooxygenase(COX) inhibitors and others

In one embodiment, the method of using the compounds, compositions andthe formulations as discussed above for treating MS is dose-dependent.For example, to achieve a better treatment of MS, 0.8, 0.85, 0.9, 0.95,1.0, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 2, 2.5, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5,12, 12.5, 13, 13.5, 14, 14.5 or 15 g/kg per body weight of homosalateand/or octisalate may be administered to a MS patient or a perspectiveMS patient.

Preferably, 1.0, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5,2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10,10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5 or 15 g/kg per body weightof homosalate and/or octisalate may be administered to a MS patient or aperspective MS patient.

FIGS. 8A, 8B, 8C and 8D show dose dependent suppression of EAE byhomosalate (HS) and octisalate (OS). FIGS. 8A and 8B: Mice were treatedwith various doses (10, 20 and 30 μl or 0.5, 1.0, 1.5 g/kg) ofhomosalate or octisalate topically. The mean score (FIG. 8A) and bodyweights (FIG. 8B) were recorded. FIGS. 8C and 8D, Mice treated withhomosalate (30 μl or 1.5 g/kg) topically at various times. Daily meanscore (FIG. 8C) and body weight (FIG. 8D) were recorded. Data areexpressed as mean value. In FIG. 8A, all treatment groups weresignificantly different from control (p<0.05). In FIG. 8B, alltreatments except homosalate 0.5 g/kg were significantly different fromcontrol (p<0.05); In FIGS. 8C and 8D, the mean scores of homosalateevery day and every 2-day were significantly lower than control(p<0.05).

Preferred Patients

A preferred patient of the present invention is an MS patient whoexhibits at least one of the following autonomic, visual, motor, orsensory symptom: loss of sensitivity or changes in sensation such astingling, numbness, muscle weakness, very pronounced reflexes, musclespasms, or difficulty in moving; difficulties with coordination andbalance (ataxia); problems with speech or swallowing, visual problems(nystagmus, optic neuritis or double vision), feeling tired, acute orchronic pain, bladder and bowel difficulties, confused thinking,emotional problems such as depression or unstable mood, Uhthoff'sphenomenon (a worsening of symptoms due to exposure to higher than usualtemperatures) or Lhermitte's sign (an electrical sensation that runsdown the back when bending the neck).

One may also wish to measure therapeutic outcome by using conventionalMS monitoring rubrics. For example, disability and severity can bemeasured by the expanded disability status scale (EDSS) or the multiplesclerosis functional composite. Magnetic resonance may also be used toshow a reduction in new lesions in the nervous system.

The definition of “symptom” includes all these parameters.

Another preferred patient of the present invention is a patient indanger of developing MS or a “prospective patient.”

Preferred patients may be pediatric patients, geriatric patients oradult patients. Preferred patients may be either male or female.

Preferred Therapeutic Compositions

The method of the present invention requires exposing a patient to octylsalicylate, homosalate or a combination of both.

Octyl salicylate; 2-ethylhexyl salicylate; 2-ethylhexyl2-hydroxybenzoate; ethyl hexyl salicylate; 2-ethylhexyl ester salicylicacid; salicylic acid, 2-ethylhexyl ester; benzoic acid, 2-hydroxy-,2-ethylhexyl ester; 2-ethylhexyl ester benzoic acid, 2-hydroxy-;2-hydroxy-2-ethylhexyl ester benzoic acid; or octisalate, is an esterformed by the condensation of a salicylic acid with 2-ethylhexanoloftenand is used as an ingredient in sun screens and cosmetics to absorb UVB(ultraviolet) rays from the sun. It is typically found as a colorlessoily liquid with a slight floral odor. Octyl salicylate can be obtainedfrom Spectrum Chemical MFG. Corp. (New Brunswick, N.J.).

The salicylate portion of the molecule absorbs ultraviolet light,protecting skin from the harmful effects of exposure to sunlight. Theethylhexanol portion is a fatty alcohol, adding emollient and oil-like(water resistant) properties.

The formula for octyl salicylate is presented below:

Homosalate, or 3,3,5-trimethylcyclohexyl 2-hydrobenzoate, is an esterformed from salicylic acid and 3,3,5-trimethylcyclohexanol, a derivativeof cyclohexanol. The compound is contained in 45% of U.S. sunscreens andis sometimes used as a chemical UV filter. The salicylic acid portion ofthe molecule absorbs ultraviolet rays with a wavelength from 295 nm to315 nm, protecting the skin from sun damage. The hydrophobiccyclohexanol portion provides greasiness that prevents it fromdissolving in water.

Homosalate can be obtained from Spectrum Chemical MFG. Corp. (NewBrunswick, N.J.).

The formula for homosalate is presented below:

Preferred Methods of Administration

The compounds may be introduced to a patient in a variety ofadministrative modes. Preferable administration may be topical,intravenous, oral, parenteral, or via inhalation. Other modes ofadministration will also be within the scope of the present invention.

The Examples below disclose an effective topical dose in a mouse modelof 1.5 g/kg each day for homosalate and 0.5 g/kg daily for octylsalicylate. The corresponding human dose would be in the range of 0.1 to5 g/kg body weight. We predict an effective dose range of 0.1 to 2 g/kgbody weight for homosalate and 0.1 to 1 g/kg body weight for octylsalicylate.

An oral, inhalation or parenteral dose will typically be similar to atopical dose.

A preferred dose will achieve an effective systemic concentration of thecompound.

Example 1

Materials and Methods

Animals and Diet. Female C57BL/6 mice (8-9 weeks old) purchased fromJackson Laboratory were fed a standard lab diet chow 5008 (Purina Mills,Richmond, Ind.) and maintained in the Department of Biochemistry'svivarium. The mice were exposed to 12 h light-dark cycles. Allprocedures were approved by the Research Animal Resources Committee ofthe College of Agricultural & Life Sciences, University ofWisconsin-Madison. The mice were randomly divided into groups fordifferent treatment (12 mice/group).

Sunscreen and Active Ingredients Treatment

Mice were shaved on their back to receive sunscreen or NBUVB radiation.The following Sunscreen cream or spray was used in our study (SeeAppendix A): 1. Coppertone spray (SPF 50, MSD Consumer Care, Inc.,Memphis, Tenn.); 2. Coppertone water babies (SPF 50, MSD Consumer Care,Inc., Memphis, Tenn.); 3. Hawaiian Tropic (SPF 50, Energizer PersonalCare, LLC, Shelton, Conn.); 4. Kiss my face (SPF 50, Kiss My Face, LLC,Gardiner, N.Y.); 5. Blue Lizard Australian Sunscreen (SPF 30, CrownLaboratories, Johnson, Tenn.); 6. Banana Boat Kids (SPF 50, EnergizerPersonal Care, LLC, Shelton, Conn.); 7. CoTZ Face (SPF40, CoTZ Skincare,West Norriton, Pa.). The sunscreen was applied manually to cover theshaved skin daily depending on the experimental schedule. Four mainactive ingredients (avobenzone; oxibenzone; homosalate; octylsalicylate) were purchased from Spectrum Chemical MFG. Corp. (NewBrunswick, N.J.). The total dose of each ingredient administratedtopically on each mouse skin was equal to the amount of the ingredientin the sunscreen.

Narrow Band UVB (NBUVB) Treatment

For NBUVB treatment, a set of four TL20W/01 UVB 311 narrow band 2 Ftbulbs (wavelength centered at 311 nm-313 nm, Amjo Corp, West Chester,Ohio) were used. The radiation output was measured by using a UVradiometer equipped with a 302-nm sensor (UVP LLC, Upland, Calif.). Micewere put into a 16-chamber Plexiglas cage individually to receive dailyUV radiation from day 7 prior to immunization to day 30 afterimmunization. Each mouse was rotated in the different chamber to avoiduneven UV radiation in the experiment.

EAE Induction

In this experiment, mice were immunized with myelin oligodendrocyteglycoprotein peptide (MOG)₃₅₋₅₅. MOG₃₅₋₅₅ kit (EK-2110) was purchasedfrom Hooke lab (Lawrence, Mass.). Each mouse was immunized withsubcutaneous injection of 20 μl MOG₃₅₋₅₅/CFA emulsion andintraperitoneally injection with 200 ng of pertussis toxin (ListBiological Laboratories) diluted in sterile PBS. The second boosterpertussis toxin injection was given 48 hours later. Each mouse wasscored daily for clinical signs of EAE using the following scale: 0, noclinical disease; 1, loss of tail tone; 2, unsteady gait; 3, hind limbparalysis; 4, forelimb paralysis; 5, death.

Statistical Analysis

Onset was calculated by averaging the first day when clinical signsappeared and continued for at least 2 days. Mean severity was determinedby averaging the clinical scores during the entire experiment. The CDI(clinical disease index) was calculated by summing the clinical scoresin each animal and divided by the number of mice per group. Statisticalanalyses were performed using the two-tailed Fisher exact probabilitytest for incidence, the Mann-Whitney nonparametric u test for clinicalscores, and the unpaired Student t test for all other measurements. Avalue of P<0.05 was considered statistically significant.

Results

FIGS. 9A, 9B, 9C, 10A, 10B, 10C, 10D, 10E and 10F show the protectiveeffect of various sun screens on mice from developing EAE, although thesunscreens protect at different levels. Testing individual sunscreeningredients demonstrate that homosalate and octyl salicylate componentsprovide the protection.

Referring to FIG. 1, our data demonstrate that topical application ofsunscreen protects mice from EAE progression. Mice received topicalapplication of sunscreen, NBUVB or both after immunization withMOG35-55/CFA emulsion. Each mouse was scored daily after EAE during theentire experiment. Data are expressed as mean value, N=10-12/group.

Referring to FIGS. 2A and 2B, we analyzed different sunscreeningredients. Avobenzone and oxybenzone partially suppress EAE severityin mice. Mice were treated with ingredients (avobenzone, oxybenzone orcombination) of sunscreen by topical administration daily during EAE(day 0-day 30). Sunscreen spray 200 μl was also used as a positivecontrol. Each mouse was scored daily and weighed weekly during theexperiment (30 days). Data are expressed as mean value, N=10-12/group.

Referring to FIGS. 3A and 3B, sunscreen ingredients (homosalate andoctyl salicylate) dramatically suppress EAE development in mice withoutaffecting body weight. Mice were treated with ingredients (homosalate,octyl salicylate or combination) of sunscreen by topical administrationdaily during EAE (day 0-day 30). Sunscreen spray 200 μl daily was usedas a positive control. Mice were scored daily and weighed weekly. Datawere expressed as mean value, N=10-12/group.

Table 1, below, summarized the effect of different sunscreen ingredientson EAE mice.

TABLE 1 Mean Treatment Incidence Day of onset Severity CDI Control 100%(12/12)   16 ± 1  2.5 ± 0.8  42 ± 1   Homosalate 8% (1/12) * 22 ± 0 *1.3 ± 0.0 * 1 ± 0 * octyl 75% (9/12)   18 ± 3 * 2.0 ± 0.8 * 20 ± 1 * salicylate Homo + 0% (0/12) *  0 ± 0 * 0.0 ± 0.0 * 0 ± 0 * OctiSunscreen 0% (0/11) *  0 ± 0 * 0.0 ± 0.0 * 0 ± 0 * Data were expressedas mean ± SD. N = 11-12; * P < 0.05 vs Control.

Example 2

Salate Derivatives Found in Sunscreens Block Experimental AutoimmuneEncephalomyelitis in Mice.

Abstract

Ultraviolet light (UV) suppresses experimental autoimmuneencephalomyelitis (EAE, an animal model of MS) in mice and may beresponsible for decreased incidence of MS in equatorial regions.

To test this concept further, we applied commercially available sunblockpreparations to mice before exposure to UV light. Surprisingly, some ofthe sunblock preparations blocked EAE without UV light. Further, varioussunblock preparations had variable ability to suppress EAE. By examiningthe components of the most effective agents, we found that homosalateand octisalate were the components responsible for suppressing EAE.Salates therefore maybe useful in stopping the progression of MS and mayprovide new insight into mechanisms of controlling autoimmune disease.

Significance

Multiple sclerosis (MS) is an autoimmune disease that is difficult tomanage and for which there is no cure. We have discovered that certainspecific sunblock preparations can prevent the development ofexperimental autoimmune encephalomyelitis (EAE), a widely used animalmodel of MS. Salate esters in the sunblock preparations were foundresponsible for preventing EAE. This suggests that the salate esters maybe of value in arresting the symptoms of MS.

Introduction

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease ofthe central nervous system (CNS), affecting 2.5 million people worldwide(1). In 1974, Agranoff and Goldberg observed that the incidence of MS isinversely related to the sun exposure in both hemispheres (2). Goldbergsuggested that increased vitamin D production caused by sunlightexposure may be responsible for the reduction of MS incidence (3).However, more recent results have made this hypothesis unlikely andinstead a narrow band of UVB (NBUVB) light at 300-315 nanometers hasbeen shown to suppress experimental autoimmune encephalomyelitis (EAE),the animal model of MS (4). This narrow band of light does not causesynthesis of Vitamin D from 7-Dehydrocholesterol (5). During the courseof our studies we used commercial sunblock preparations presumably toprevent the suppression of EAE by NBUVB. Quite unexpectedly somesunblock preparations themselves completely prevented EAE without UVlight. Further, commercial sunblock preparations varied widely in theirability to suppress EAE. By examining the components of thepreparations, we found that two components were responsible forsuppression of EAE. These two components are esters of salicylic acidi.e. homosalate and octisalate. We now report these findings.

Results

Topical Application of Sunscreen (Coppertone Spray) CompletelySuppressed EAE Development.

When mice were treated with NBUVB, the average disease severity wasdramatically decreased similar to previous studies (FIG. 4A). Sunscreenapplication prior to NBUVB did not prevent the suppression by NBUVB(FIGS. 4A and 4C). The body weight changes observed in these animals wasconsistent in the disease outcome (FIG. 4B). Surprisingly, topicaladministration of sunscreen itself completely blocked EAE (FIG. 4C).

Commercial Sunscreens Differ Markedly in their Ability to Suppress EAE.

When 6 brands of sunscreen were tested on EAE, only 4 brands (HAWAIIANTROPIC, COPPERTONE, KISS MY FACE, BLUE LIZARD) produced a significantsuppression (FIG. 5A), the remaining two brands of sunscreen (BANANABOAT, COTZ FACE) were without effect (FIG. 5A). To be sure that theeffect of the sunscreen simply did not prevent a total block of allwavelengths, total darkness (−7 to 30 days) was tested and was withouteffect on EAE regardless of when it was initiated (FIG. 5B).Interestingly, additional testing indicated that sunscreen applicationwas necessary at the time of immunization (FIG. 6A). The body weightswere slightly but significantly higher in mice receiving treatment withsunscreen either prior to or at the time of immunization (FIG. 6B). Thesuppression of disease by sunscreen was dose-dependent (FIG. 6C) andincreased body weights correlated with the improvement of disease (FIG.6D).

Homosalate and Octisalate were the Ingredients that Caused EAESuppression; Avobenzone and Oxybenzone Produced Only Slight Suppressionof EAE.

A dose of 30 μl homosalate (1.5 g/kg) and 10 μl octisalate (0.5 g/kg)were calculated to be the amount delivered by Coppertone Spraysunscreen. This quantity was applied to the mice. Sunscreen spray at 200μl (containing homosalate 15%, octisalate 5%) was applied to EAE mice asa positive control. The results showed that homosalate only and acombination of homosalate and octisalate dramatically suppressed EAEseverity. (FIG. 7C and Table 1). Octisalate at the dose of 0.5 g/kgproduced a moderate suppression but did not reach statisticalsignificance (FIG. 7C and Table 1). The degree of the disease wasreflected in body weight (FIG. 7D). The other two ingredients(avobenzone and oxybenzone) and the combination produced little to nosignificant suppression of EAE (FIG. 7A) and no significant change inbody weight (FIG. 7B).

Homosalate and Octisalate Suppression was Dose Dependent.

When tested at three different doses (0.5, 1.0, 1.5 g/kg), bothhomosalate and octisalate exhibited dose-dependent suppression of EAE(FIG. 8A). With the exception of the lowest dose of homosalate, alltreatments increased the body weight (FIG. 8B). When homosalate wasapplied less frequently than each day, its effectiveness diminished(FIGS. 8C and 8D).

Discussion

Because UV light and especially narrow band UV light can suppress EAE,it was surprising to find that sunblock creams could prevent thedevelopment of EAE even in the absence of UV light. On closerexamination it became clear that not all sun block preparations possessthis property. Further, complete absence of light was without effect onEAE. We thus focused on the component(s) of the active sunblockpreparations. The suppression of EAE by the active sunblock preparationswas traced to two salate esters i.e. homosalate and octasalate. Whentested directly both salates were equally active at ca 1.5 g/kg insuppressing EAE. It is likely that these compounds are not acting byblocking or absorbing UV light. Simply keeping mice in a completeabsence of light did not affect the development of EAE. Further, somesunscreens that are effective as sun blockers do not suppress EAE. Onlysunblocks that contain salate esters are effective and the salatesthemselves clearly block EAE. The only adverse effect of the homosalateand octisalate is a temporary mild skin irritation. A topical dose of1.5 g/kg of homosalate, which completely blocked EAE, is below theunpublished acute dermal toxicity (LD50>5 g/kg for rabbits).

The complete suppression of EAE by topical administration of homosalateand octisalate is a novel finding. Salicylates are well-knownnonsteroidal anti-inflammatory drugs (NSAIDs)(6). The anti-inflammatoryeffect of homosalate has been demonstrated in another study by using theear edema test in mice (7). A related compound, aspirin (acetylsalicylicacid, ASA) is a traditional anti-inflammatory pharmaceutical (8). Therelationship between aspirin and MS was studied in 1961 without asignificant difference between the control and treated group (9). Therehas not been a direct study of aspirin and MS development. Inhibition ofcyclooxygenase (COX) is the primary mechanism of NSAID (10). COX-2 hasbeen observed in MS lesions (11). Recently, cyclooxygenase (COX)inhibitors, have shown significant suppression of EAE (12, 13). It isclear that we have no explanation of how the salates prevent EAE.However, this finding presents a clear opportunity to explore not onlymechanisms but also new approaches to therapy of MS.

Materials and Methods

Animal Husbandry.

Female C57BL/6 mice (8-10 weeks old) purchased from Jackson Laboratory(Bar Harbor, Me.) were fed a standard lab chow 5008 (Purina Mills,Richmond, Ind.). The mice were exposed to 12 h light-dark cycles. In oneexperiment the animals were kept in darkness at all times. Allprocedures were approved by the Institutional Animal Care and UseCommittee of the College of Agricultural & Life Sciences, University ofWisconsin-Madison.

EAE Induction

Mice were immunized with MOG₃₅₋₅₅ kit (EK-2110, purchased from Hooke lab(Lawrence, Mass.)). Each mouse was immunized with a subcutaneousinjection of 20 μl MOG₃₅₋₅₅/CFA emulsion and an intraperitonealinjection with 200 ng of pertussis toxin (List Biological Laboratories)diluted in sterile PBS (14). A second booster pertussis toxin injectionwas given 48 hours later. Each mouse was scored daily for clinical signsof EAE using the following scale: 0, no clinical disease; 1, loss oftail tone; 2, unsteady gait; 3, hindlimb paralysis; 4, forelimbparalysis; 5, death (15).

Sunscreens and Active Ingredients Treatment

Sunscreen creams or spray applied topically to the shaved back skin ofthe mice daily are shown below (Table 2): 1. BANANA BOAT KIDS(SPF 50,Energizer Personal Care, LLC, Shelton, Conn.); 2. BLUE LIZARD AUSTRALIANSUNSCREEN (SPF 30, Crown Laboratories, Johnson, Tenn.) 3. COPPERTONESPRAY (SPF 50, MSD Consumer Care, Inc., Memphis, Tenn.); 4. COTZ FACE(SPF40, CoTZ Skincare, West Norriton, Pa.); 5. HAWAIIAN TROPIC (SPF 50,Energizer Personal Care, LLC, Shelton, Conn.); 6. KISS MY FACE (SPF 50,Kiss My Face, LLC, Gardiner, N.Y.). Each sunscreen was applied daily tocover the shaved skin (100-200 μl). Avobenzone, oxibenzone, homosalate,and octisalate were purchased from Spectrum Chemical Mfg. Corp. (NewBrunswick, N.J.). Avobenzone and oxibenzone were dissolved in DMSO.Homosalate and octisalate were applied directly. The total dose of eachingredient administrated topically on each mouse skin was equal to theamount provided by the respective sunscreen.

TABLE 2 Sunscreen brands tested. Trade name SPF Active IngredientsBanana Boat 50 Titanium dioxide 3.1%, Zinc oxide 4.0% Kids Blue Lizard30 Octinoxate 7.5%, Octicrylen 2.0%, Oxybenzone 3.0%, Zinc oxide 6.0%Coppertone 50 Avobenzone 3.0%, Homosalate 15.0%, Octisalate (Spray) 5.0%CoTZ Face 40 Titanium dioxide 8.0%, Zinc oxide 3.8% Hawaiian 50Avobenzone 2.7%, Homosalate 8.0%, Octisalate Tropic 4.5%, Octocrylen5.0% Kiss My Face 50 Avobenzone 4.0%, Homosalate 5.0%, Octinoxate 7.5%,Octisalate 5.0%, Zinc oxide 1.7%

Narrow Band UVB (NBUVB) Treatment

For NBUVB treatment, a set of four TL20W/01 UVB 311 narrow band 2 ft.bulbs (wavelength centered at 311-313 nm, Amjo Corp, West Chester, Ohio)were used daily at (10 KJ/m²) (14). The radiation output was measuredusing a UV radiometer equipped with a 302-nm sensor (UVP LLC, Upland,Calif.). A 16-chamber Plexiglass cage was used for daily UV radiation.Each chamber contained one mouse. The mice were rotated through thedifferent chambers so that each mouse received equal light exposure. Themice were UV radiated beginning at the same day of immunizationcontinuing through 30 days post immunization.

REFERENCES

-   1. Compston A & Coles A (2002) Multiple sclerosis. Lancet (London,    England) 359(9313):1221-1231.-   2. Agranoff B W & Goldberg D (1974) Diet and the geographical    distribution of multiple sclerosis. Lancet (London, England)    2(7888):1061-1066.-   3. Goldberg P (1974) Multiple sclerosis: vitamin D and calcium as    environmental determinants of prevalence. International Journal of    Environmental Studies 6(1):19-27.-   4. Wang Y, et al. (2013) Suppression of experimental autoimmune    encephalomyelitis by 300-315 nm ultraviolet light. Archives of    biochemistry and biophysics 536(1):81-86.-   5. MacLaughlin J A, Anderson R R, & Holick M F (1982) Spectral    character of sunlight modulates photosynthesis of previtamin D3 and    its photoisomers in human skin. Science (New York, N.Y.)    216(4549):1001-1003.-   6. Paulus H E & Whitehouse M W (1973) Nonsteroid anti-inflammatory    agents. Annual review of pharmacology 13:107-125.-   7. Couteau C, Chauvet C, Paparis E, & Coiffard L (2012) UV filters,    ingredients with a recognized anti-inflammatory effect. PloS one    7(12):e46187.-   8. Tsau S, Emerson M R, Lynch S G, & LeVine S M (2015) Aspirin and    multiple sclerosis. BMC medicine 13:153.-   9. Miller H, Newell D J, & Ridley A (1961) Multiple sclerosis.    Trials of maintenance treatment with prednisolone and soluble    aspirin. Lancet (London, England) 1(7169): 127-129.-   10. Farah A E & Rosenberg F (1980) Potential therapeutic    applications of aspirin and other cyclo-oxygenase inhibitors.    British journal of clinical pharmacology 10 Suppl 2:261s-278s.-   11. Rose J W, Hill K E, Watt H E, & Carlson N G (2004) Inflammatory    cell expression of cyclooxygenase-2 in the multiple sclerosis    lesion. Journal of neuroimmunology 149(1-2):40-49.-   12. Miyamoto K, et al. (2006) Selective COX-2 inhibitor celecoxib    prevents experimental autoimmune encephalomyelitis through    COX-2-independent pathway. Brain: a journal of neurology 129(Pt    8):1984-1992.-   13. Marusic S, et al. (2008) Blockade of cytosolic phospholipase A2    alpha prevents experimental autoimmune encephalomyelitis and    diminishes development of Th1 and Th17 responses. Journal of    neuroimmunology 204(1-2):29-37.-   14. Wang Y, Marling S J, Martino V M, Prahl J M, & Deluca H F (2016)    The absence of 25-hydroxyvitamin D3-1alpha-hydroxylase potentiates    the suppression of EAE in mice by ultraviolet light. The Journal of    steroid biochemistry and molecular biology.-   15. Becklund B R, Severson K S, Vang S V, & DeLuca H F (2010) UV    radiation suppresses experimental autoimmune encephalomyelitis    independent of vitamin D production. Proceedings of the National    Academy of Sciences of the United States of America    107(14):6418-6423.

The invention claimed is:
 1. A method of treating a prospective multiplesclerosis (MS) patient, comprising the steps of: (a) Identifying aprospective MS patient, and (b) Treating the patient with an effectiveamount of a composition selected from the group consisting ofhomosalate, octyl salicylate and combinations of homosalate and octylsalicylate, wherein occurrence of MS symptom is lessened or progressionof the symptom is slowed or stalled.
 2. The method of claim 1, whereinthe symptom is muscle weakness/numbness.
 3. The method of claim 1,wherein the treatment is daily.
 4. The method of claim 1, wherein thetreatment is at least 30 days in duration.
 5. The method of claim 1,wherein the composition is delivered as an oral dose.
 6. The method ofclaim 1, wherein the composition is delivered as a topical dose.
 7. Themethod of claim 1, wherein the composition is delivered as a parenteraldose.
 8. The method of claim 1, wherein the composition compriseshomosalate or the combination of homosalate and octyl salicylate.
 9. Themethod of claim 1, wherein the compositions consist essentially ofhomosalate.
 10. The method of claim 1, wherein the composition consistsessentially of the combination of homosalate and octyl salicylate.